br Materials and methods br Results br Discussion
Materials and methods
Discussion Wound healing of the human cornea is a complex process that requires cell migration and proliferation. Corneal wound healing is characterized by major changes in the composition of the ECM such as a massive transitory secretion of fibronectin combined with a reduction in the secretion of collagens and laminins , , . This ECM remodeling is required to allow fast migration of corneal epithelial cells in order to rapidly cover the damaged area. In the present study, we used the hTEC as a model to study the signal transduction pathways, and their participating protein mediators, that are involved in corneal wound closure. We demonstrated that expression of a few protein mediators from both the PI3K/Akt and MAPK pathways was altered during wound healing of damaged hTECs. Increased phosphorylation of Akt combined to a reduced phosphorylation of the CREB mediators were among the most striking alterations observed. By supplementing the culture medium of wounded hTECs with both the CREB inhibitor C646 and the Akt agonist SC79, we could considerably accelerate wound closure in our model. A few human models of corneal wound healing have been described , , . Ex vivo organ culture models ,  take advantage of the native tissue but the limited availability and inter-individual variability restrict the number of analyses that can be performed simultaneously. In contrast, a large number of conditions and amount of treated cells is possible with the scratch assay. However, epithelial cells are in contact with the growth medium that contains numerous supplements and growth factors since HCECs are cultured submerged in the culture medium. The hTEC has the advantage of a 3D structure that renders possible the culture at the air–liquid interface. Under this condition, the medium containing the growth factors is not in direct contact with epithelial cells since it is only present under the reconstructed stroma. Consequently, the epithelium is nourished indirectly, a condition that better mimic the in vivo condition seen with the native cornea. Our gene profiling analyses revealed alterations in the expression of a few genes coding for mediators from both the MAPK and PI3K/Akt pathways, including genes, such as PTK2 and members of the Akt family, whose expression is increased in the central wounds, whereas others, such as CREB, are decreased in wounded hTECs. PTK2 encodes for the kinase FAK (focal adhesion kinase) that is activated through a yet poorly understood mechanism possibly involving FAK clustering, autophosphorylation of FAK at Y397  and a mechanical linkage of integrins to the xanthine oxidase inhibitor cytoskeleton . This increased expression of PTK2 is also consistent with the coordinated increase in the expression of the β1-associated integrins α1, α5, α6 and α11 that we also observed in our wounded hTECs (Supplementary Fig. 3). Under normal circumstances, activation of FAK leads to the downstream activation of the ERK kinases , a required step in the activation of a number of transcription factors such as c-Jun , AP-1, NFkB, Nrf2  and Sp1 . However, our results demonstrate clearly that phosphorylation of ERK kinases is considerably reduced (more than 18-fold reduction) in the central wound relative to its level in the external area of wounded hTECs. We previously reported that expression of the α5, α6 and α9 integrin subunits were negatively regulated by the transcription factor NFI , , . Interestingly, Zheng et al. reported that expression of the NFI isoform NFIA was induced in an ERK-dependent manner in mouse neurons . This raises the interesting possibility that the increased expression observed for both the α5 and α6 integrin genes in our wounded hTECs may result, at least in part, from the coordinated reduction in NFI activity in response to a reduction in ERK activation. The kinase arrays conducted in this study demonstrated that both the activation of Akt and the suppression of the CREB activity are required in order to ensure proper wound closure of punch-damaged hTECs. Phosphorylated Akt as being involved in wound healing is no surprise. Akt, also referred to as protein kinase B, is a serine threonine kinase that plays many critical roles in biological processes such as cell survival, proliferation, migration, angiogenesis and metabolism. It is activated downstream of phosphatidylinositol 3-kinase (PI3K)  by a variety of growth factors and cytokines. The upregulated levels of Akt that have been reported in exaggerated wound-healing responses , together with decreased Akt signaling in chronic, non-healing wounds  all point to the central role played by Akt in wound healing. Indeed, Xiao et al. recently demonstrated that treatment of dorsal skin wounded C57BL/6 mice with ozone oil facilitated the wound healing by increasing fibroblast migration via PI3K/Akt/mTOR signaling pathway in vivo. Similar observations were also reported for wound healing of the rat skin, the impaired wound healing observed for the skin of diabetic rats being reported to result from a dysfunction in the Akt/mTOR pathway . Most interestingly, high glucose was reported to suppress Akt phosphorylation in a ROS sensitive manner, a process that also delayed corneal epithelial wound healing in the porcine cornea . On the other hand, much less is known about the impact CREB phosphorylation has on wound healing. We initially thought that the negative action of phosphorylated CREB on HCECs proliferation in wounded hTECs could rely on its ability to increase the expression of the antiproliferative molecule p21 as target sites for CREB have been identified in the promoter region of the p21 gene . However, close comparison of the microarray data between the external region and central wound revealed no significant variation in the expression of the p21 gene (Supplementary Table 2). Somehow consistent with our results, CREB phosphorylated at its serine 133 residue has been recently found to act as a negative regulator of PDGF-BB-stimulated vascular smooth muscle cells (VSMCs) proliferation . Similarly, a migration-inhibitory function for CREB was also reported in glioma cells, a process that apparently depends on a CREB-miR-9 negative feedback minicircuitry . Interestingly, CREB was found to simultaneously play a pro-proliferative and anti-migratory role in such cells, a process described by these authors as the “go or grow” process. Suppression of CREB by miR-150, that also demonstrates the ability to also suppress expression of EP300, was reported to increase migration and invasiveness of HCT116 cells, a mechanism that could also be reproduced by knocking-down CREB expression by CRISPR/Cas9 . However, and to our knowledge, we are the first to demonstrate that both these mechanisms (suppression and activation of CREB and Akt activities, respectively) occur simultaneously while wound healing of hTECs is proceeding.